This project focuses on the development of fumagillin analogs to combat failures of standard care antigiardiasis and antiamebiasis drugs. Giardia lamblia and Entamoeba histolytica are highly infective parasites that cause severe diarrheal diseases, leading to much suffering in poverty stricken regions worldwide. All standard care drugs have undesirable side effect, giardiasis treatment fails at a frequency of ~20% cases, and there are only a few drugs available to treat amebiasis for patients that do not tolerate the side effects. Giardia drug resistance has become a concern, and G. lamblia and E. histolytica drug-resistant strains can be readily raised in the laboratory. The arsenal of drugs available for treating giardiasis and amebiasis belong to only a few chemical classes, primarily nitroimidazoles and thiazolides. Because G. lamblia drug resistance already exists for these classes of compounds, it is likely to progress rapidly to new class members. Thus, new drug development projects should focus on identifying new chemical scaffolds. Using compound screening of an approved drug library, we discovered that fumagillin, an orphan drug used in the European Union to treat intestinal microsporidiosis in immune compromised patients, kills Giardia trophozoites in vitro with high potency and exhibits superior efficacy in infected mice compared with the standard care drug, metronidazole. Moreover, 60 years ago, fumagillin was reported to effectively cure human intestinal amebiasis. Fumagillin is a methionine aminopeptidase 2 inhibitor, an enzyme that has not been targeted yet for antigiardiasis and antiamebiasis drug development. While fumagillin is expected to be effective in curing both giardiasis and amebiasis it has two liabilities, potential toxicity and heat and humidity instabiliy. To optimize drug properties, we will synthesize fumagillin derivatives that are less liable to chemical instability on storage and metabolic degradation, and concomitantly have reduced permeability through the intestinal epithelial barrier to prevent potential toxic effects. New methionine aminopeptidase 2 inhibitors that will show good potency in metronidazole-responsive and metronidazole-resistant G. lamblia and/or E. histolytica strains as well as provide increased stability and reduced permeability across Caco-2 monolayers will be investigated in vivo in giardiasis and amebiasis mouse models. The pharmacokinetics and toxicity profiles of the most efficacious compounds will be determined in mice to confirm that the reduced in vitro permeability leads to reduced host bioavailability and toxicity in vivo. The outcome of the project will be 1-2 compounds efficacious against giardiasis and/or amebiasis with superior toxicity and stability properties compared with the parent drug, fumagillin. These compounds will be ready for IND-enabling studies.